Journal of Steroid Biochemistry & Molecular Biology 120 (2010) 1–10 Contents lists available at ScienceDirect Journal of Steroid Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/jsbmb Cloning, expression and enzyme activity analysis of testicular 11-hydroxysteroid dehydrogenase during seasonal cycle and after hCG induction in air-breathing catfish Clarias gariepinus  M.K. Rasheeda a , H. Kagawa b , R. Kirubagaran c , A. Dutta-Gupta a , B. Senthilkumaran a,∗ a Department of Animal Sciences, School of Life Sciences-Centre for Advanced Studies, University of Hyderabad, P. O. Central University, Hyderabad 500 046, Andhra Pradesh, India b Department of Biological Production and Environmental Sciences, Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan c National Institute of Ocean Technology, Pallikarani, Chennai, Tamil Nadu 600 100, India article info Article history: Received 8 September 2009 Received in revised form 12 February 2010 Accepted 18 February 2010 Keywords: 11-Hydroxysteroid dehydrogenase 11-Ketotestosterone Ontogeny Testicular cycle Spermatogenesis abstract A full-length cDNA encoding 11-hydroxysteroid dehydrogenase type 2 (11ˇ-HSD2) was cloned from testis of air-breathing catfish, Clarias gariepinus which showed high sequence homology to zebrafish and eel. The open reading frame of 11ˇ-HSD2 was then transfected to COS-7 cells, which converted 11-hydroxytestosterone (11-OHT) to 11-ketotestosterone (11-KT). Using NAD + , 11ˇ-HSD2 from tes- ticular microsomes oxidized 11-OHT with apparent K m 56 ± 4 nM and V max 55 ± 6 pmol/h/mg protein values. Tissue distribution analysis revealed prominent expression in testis, anterior kidney, liver and gills. Expression of 11ˇ-HSD2 in testis and serum levels of 11-KT were high in the prespawning phase. Administration of human chorionic gonadotropin (hCG) during prespawning and resting phases revealed initial rise in 11ˇ-HSD2 transcript at 4 h followed by gradual increase at 8 h, 12 h and peaking at 24 h, only in testis of prespawning phase. Rate of conversion of 11-OHT to 11-KT by testicular microsomes during different testicular phases and after hCG administration corroborated well with the expression of 11ˇ-HSD2. Ontogeny study indicated that this enzyme is expressed during testicular development. Thus the spatio-temporal expression supported with putative dehydrogenase activity and circulating 11-KT levels clearly suggest a major role for 11ˇ-HSD2 during testicular differentiation and seasonal testicular cycle in catfish. © 2010 Elsevier Ltd. All rights reserved. 1. Introduction It is widely accepted that the sex steroids are involved during the process of sex differentiation, gametogenesis and sex reversal in fish [1]. The role of estradiol-17 (the steroid hormone produced by aromatase) in ovarian differentiation, oogenesis and as a fem- inizing agent is well documented in many fish species [2–4]. The role of 11-oxygenated androgen, 11-ketotestosterone (11-KT) in teleostean male reproduction is in its primitive stage with few stud- ies suggesting its role during testis formation and differentiation [5,6], sex change in sequential hermaphrodites [7], spermatoge- nesis and sperm maturation [8,9]. On the contrary, there are also reports [10,11] which state that 11-hydroxylase (11ˇ-H), a penul- timate steroidogenic enzyme involved in the biosynthesis of 11-KT, is not expressed at early stages of testis development or dur-  This work has been done at the Department of Animal Sciences, University of Hyderabad. ∗ Corresponding author. Tel.: +91 40 23134562; fax: +91 40 23010307/120. E-mail addresses: bsksl@uohyd.ernet.in, senthilkumaranb@yahoo.com (B. Senthilkumaran). ing male sex determination. Judging from the role of 11-KT, the expression and dehydrogenase activity of 11ˇ-HSD2 (the enzyme involved in 11-KT production) might be important for testicu- lar differentiation [7]. Thus, the involvement of 11ˇ-HSD2 as a marker for testis determination in teleosts is a contentious topic and needs further investigation. We have chosen an air-breathing, gonochoristic, male heterogametic annual breeding catfish, Clar- ias gariepinus having lobular testis with synchronous developing cyst as our experimental model because of the ease in breed- ing, rearing and maintaining them in laboratory/natural (out-door tanks) conditions. These features allow us to obtain catfish lar- vae from day one until they mature to perform an ontogeny study and to determine seasonal expression and activity of 11ˇ-HSD2. Production of 11-KT can also be influenced by peripheral conver- sion more specifically from liver and anterior kidney. However, the contribution from testis cannot be ruled out as studies from our lab- oratory showed the presence of 11-KT in testis which underwent changes during thyroid hormone depletion [12,13] leading to the impairment of testicular recrudescence. Further judging from the presence of 11ˇ-HSD2 transcript and activity analysis in the testis of few teleost species [14,15], 11-KT production in testis might be essential for testicular function. This gene is primarily implicated 0960-0760/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2010.02.014